UBE2D2 is not involved in MuRF1-dependent muscle wasting during hindlimb suspension

The Ubiquitin Proteasome System (UPS) is mainly responsible for the increased protein breakdown observed in muscle wasting. The E3 ligase MuRF1 is so far the only enzyme known to direct the main contractile proteins for degradation (i.e. troponin I, myosin heavy chains and actin). However, MuRF1 does not possess any catalytic activity and thus depends on the presence of a dedicated E2 for catalyzing the covalent binding of polyubiquitin (polyUb) chains on the substrates. The E2 enzymes belonging to the UBE2D family are commonly used for in vitro ubiquitination assays but no experimental data suggesting their physiological role as bona fide MuRF1-interacting E2 enzymes are available. In this work, we first found that the mRNA levels of critical E3 enzymes implicated in the atrophying program (MuRF1, MAFbx, Nedd4 and to a lesser extent Mdm2) are tightly and rapidly controlled during the atrophy (up regulation) and recovery (down regulation) phases in the soleus muscle from hindlimb suspended rats. By contrast, E3 ligases (Ozz, ASB2β and E4b) implicated in other processes (muscle development or regeneration) poorly responded to atrophy and recovery. UBE2B, an E2 enzyme systematically up regulated in various catabolic situations, was controlled at the mRNA levels like the E3s implicated in the atrophying process. By contrast, UBE2D2 was progressively repressed during atrophy and recovery, which makes it a poor candidate for a role during muscle atrophy. In addition, UBE2D2 did not exhibit any affinity with MuRF1 using either yeast two-hybrid or Surface Plasmon Resonance (SPR) approaches. Finally, UBE2D2 was unable to promote the degradation of the MuRF1 substrate α-actin in HEK293T cells, suggesting that no functional interaction exists between these enzymes within a cellular context. Altogether, our data strongly suggest that UBE2D2 is not the cognate ubiquitinating enzyme for MuRF1 and that peculiar properties of UBE2D enzymes may have biased in vitro ubiquitination assays.     

Upregulation of MuRF1 and MAFbx participates to muscle wasting upon gentamicin-induced acute kidney injury

Acute Kidney Injury (AKI) is frequently encountered in hospitalized patients where it is associated with increased mortality and morbidity notably affecting muscle wasting. Increased protein degradation has been shown to be the main actor of AKI-induced muscle atrophy, but the proteolytic pathways involved are poorly known. The Ubiquitin Proteasome System (UPS) is almost systematically activated in various catabolic situations, and the E3 ligases MuRF1 and MAFbx are generally up regulated in atrophying muscles. We hypothesized that the UPS may be one of the main actors in catabolic skeletal muscles from AKI animals. We used gentamicin-induced acute kidney disease (G-AKI) in rats fed a high protein diet to promote acidosis. We first addressed the impact of G-AKI in the development of mild catabolic conditions. We found that both muscle atrophy and UPS activation were induced with the development of G-AKI. In addition, the phasic muscles were more sensitive to 7-days G-AKI (−11 to −17%, P < 0.05) than the antigravity soleus muscle (−11%, NS), indicating a differential impact of AKI in the musculature. We observed an increased expression of the muscle-specific E3 ligases MuRF1 and MAFbx in phasic muscles that was highly correlated to the G-AKI severity (R² = 0.64, P < 0.01 and R² = 0.71, P < 0.005 respectively). Conversely, we observed no variation in the expression of three other E3 ligases (Nedd4, Trim32 and Fbxo30/MUSA1). Altogether, our data indicate that MuRF1 and MAFbx are sensitive markers and potential targets to prevent muscle atrophy during G-AKI.     

Ets1-Mediated Acetylation of FoxO1 Is Critical for Gluconeogenesis Regulation during Feed-Fast Cycles

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高表达FoxO1抑制猪骨骼肌成肌细胞的分化

FoxO1(Forkhead box O1)是调控肌肉生长、代谢和细胞分化的重要转录因子,但其在成肌细胞分化中的作用还不甚清楚。为了研究FoxO1对哺乳动物成肌细胞分化的影响,以原代培养的长白仔猪成肌细胞作为实验材料,用2%马血清诱导分化,采用实时荧光定量PCR、Western blotting和脂质体转染等方法检测FoxO1及早期和晚期生肌调节因子MyoD和myogenin在猪成肌细胞分化过程中的表达变化。结果显示,在猪成肌细胞分化过程中,FoxO1mRNA表达量显著增加,但总蛋白量变化不显著,其磷酸化水平显著上调。同时,高表达FoxO1的猪成肌细胞中,生肌调节因子MyoD和myogenin mRNA表达受到显著抑制,而MyoD蛋白变化不显著,myogenin却显著下调(P0.05)。以上结果表明,FoxO1能够推迟猪成肌细胞的分化时间并抑制分化;同时推测,FoxO1可能通过抑制生肌调节因子的表达控制骨骼肌纤维类型的终末分化。     

Cooperative control of striated muscle mass and metabolism by MuRF1 and MuRF2

The muscle-specific RING finger proteins MuRF1 and MuRF2 have been proposed to regulate protein degradation and gene expression in muscle tissues. We have tested the in vivo roles of MuRF1 and MuRF2 for muscle metabolism by using knockout (KO) mouse models. Single MuRF1 and MuRF2 KO mice are healthy and have normal muscles. Double knockout (dKO) mice obtained by the inactivation of all four MuRF1 and MuRF2 alleles developed extreme cardiac and milder skeletal muscle hypertrophy. Muscle hypertrophy in dKO mice was maintained throughout the murine life span and was associated with chronically activated muscle protein synthesis. During ageing (months 4-18), skeletal muscle mass remained stable, whereas body fat content did not increase in dKO mice as compared with wild-type controls. Other catabolic factors such as MAFbox/atrogin1 were expressed at normal levels and did not respond to or prevent muscle hypertrophy in dKO mice. Thus, combined inhibition of MuRF1/MuRF2 could provide a potent strategy to stimulate striated muscles anabolically and to protect muscles from sarcopenia during ageing.     

Loss of ovarian function in mice results in abrogated skeletal muscle PPARδ and FoxO1-mediated gene expression

Menopause, the age-related loss of ovarian hormone production, promotes increased adiposity and associated metabolic pathology, but molecular mechanisms remain unclear. We previously reported that estrogen increases skeletal muscle PPARδ expression in vivo, and transgenic mice overexpressing muscle-specific PPARδ are reportedly protected from diet-induced obesity. We thus hypothesized that obesity observed in ovariectomized mice, a model of menopause, may result in part from abrogated expression of muscle PPARδ and/or downstream mediators such as FoxO1. To test this hypothesis, we ovariectomized (OVX) or sham-ovariectomized (SHM) 10-week old female C57Bl/6J mice, and subsequently harvested quadriceps muscles 12 weeks later for gene expression studies. Compared to SHM, muscle from OVX mice displayed significantly decreased expression of PPARδ (3.4-fold), FoxO1 (4.5-fold), PDK-4 (2.3-fold), and UCP-2 (1.8-fold). Consistent with studies indicating PPARδ and FoxO1 regulate muscle fiber type, we observed dramatic OVX-specific decreases in slow isoforms of the contractile proteins myosin light chain (11.1-fold) and troponin C (11.8-fold). In addition, muscles from OVX mice expressed 57% less myogenin (drives type I fiber formation), 2-fold more MyoD (drives type II fiber formation), and 1.6-fold less musclin (produced exclusively by type II fibers) than SHM, collectively suggesting a shift towards less type I oxidative fibers. Finally, and consistent with changes in PPARδ and FoxO1 activity, we observed decreased expression of atrogin-1 (2.3-fold) and MuRF-1 (1.9-fold) in OVX mice. In conclusion, muscles from ovariectomized mice display decreased PPARδ and FoxO1 expression, abrogated expression of downstream targets involved in lipid and protein metabolism, and gene expression profiles indicating less type I oxidative fibers.     

Double-strand breaks trigger MRX- and Mec1-dependent, but Tel1-independent, checkpoint activation

Together with the Tel1 PI3 kinase, the Mre11/Rad50/Xrs2 (MRX) complex is involved in checkpoint activation in response to double-strand breaks (DSBs), a function also conserved in human cells by Mre11/Rad50/Nbs1 acting with ATM. It has been proposed that the yeast Tel1/MRX pathway is activated in the presence of DSBs that cannot be resected. The Mec1 PI3 kinase, by contrast, would be involved in detecting breaks that can be processed. The significance of a Mec1/MRX DSB-activated DNA damage checkpoint has yet to be reported. To understand whether the MRX complex works specifically with Tel1 or Mec1, we investigated MRX function in checkpoint activation in response to endonuclease-induced DSBs in synchronized cells. We found that the expression of EcoRI activated the G1 and intra-S phase checkpoints in a MRX- and Mec1-dependent, but Tel1-independent manner. The pathways identified here are therefore different from the Tel1/MRX pathway that was previously reported. Thus, our results demonstrate that MRX can function in concert with both Mec1 and Tel1 PI3K-like kinases to trigger checkpoint activation in response to DSBs. Importantly, we also describe a novel MRX-independent checkpoint that is activated in late S-phase when cells replicate their DNA in the presence of DSBs. The existence of this novel mode of checkpoint activation explains why several previous studies had reported that mutations in the MRX complex did not abrogate DSB-induced checkpoint activation in asynchronous cells.     

Malabaricone C inhibits PDGF-induced proliferation and migration of aortic smooth muscle cells through induction of heme oxygenase-1

Malabaricone C (Mal-C), isolated from nutmeg, is known to exert a variety of pharmacological activities. However, the effect of Mal-C on vascular smooth muscle cells (VSMCs) is unknown. This study examined the effect of Mal-C on proliferation and migration of primary rat aortic smooth muscle cells (RASMCs) as well as its underlying mechanisms. Treatment of RASMCs with Mal-C induced both protein and mRNA expression of heme oxygenase-1 (HO-1) in a dose- and time-dependent manner. Mal-C-mediated HO-1 induction was inhibited by treatment with actinomycin D or by cycloheximide. SB203580 (a p38 inhibitor), SP600125 (a JNK inhibitor), U0126 (a MEK inhibitor), and N-acetylcysteine (NAC, an antioxidant) did not suppress Mal-C-induced HO-1 expression. In contrast, LY294002 (a PI3K inhibitor) blocked Mal-C-induced HO-1 expression. Moreover, RASMCs treated with Mal-C exhibited activation of AKT in a dose- and time-dependent manner. Treatment of RASMCs with Mal-C increased nuclear translocation of nuclear factor-E2-related factor 2 (Nrf2), which is a key regulator of HO-1 expression, and this translocation was also inhibited by LY294002. Consistent with the notion that HO-1 has protective effects against VSMCs, Mal-C remarkably inhibited platelet-derived growth factor (PDGF)-induced proliferation and migration of RASMCs. However, inhibition of HO-1 significantly attenuated the inhibitory effects of Mal-C on PDGF-induced proliferation and migration of RASMCs. Taken together, these findings suggest that Mal-C could suppress PDGF-induced proliferation and migration of RASMCs through Nrf2 activation and subsequent HO-1 induction via the PI3K/AKT pathway, and may be a potential HO-1 inducer for preventing or treating vascular diseases.